The Basic Skills of the Biotechnology Workplace

1. The Basic Skills of the Biotechnology Workplace Chapter 3

2. Learning Outcomes • Determine the most appropriate tool for measuring specific volumes of masses • Describe how to select, set, and use a variety of micropipets within their designated ranges to accurately measure small volumes • Convert between units of measure using the B <- -> S rule and appropriate conversion factors • Recognize the different expressions for units of concentration measurements and use their corresponding equations to calculate the amount of solute needed to make a specified solution • Define the term buffer and calculate a specified dilution from a concentrated stock solution or buffer

3. 3.1 Measuring Volumes in a Biotechnology Facility • Volume is a measurement of the amount of space something occupies • Volume is measured in • Liters (L) • Milliliters (mL) • Microliters (mL) • Different tools are used to measure volume • Graduated cylinder • Pipet • Micropipet

4. Converting Units • Often volumes are measured in one unit of measurement and reported in another • Converting between metric units • Conversion factor • To measure volumes larger than 10 milliliters, technicians usually use a graduated cylinder

5. Reading a graduated cylinder. Before using a graduated cylinder, make sure you know the total volume it will hold and the value of each of the graduations. In the lab, common graduated cylinders include 10 mL, 25 mL, 100 mL, 250 mL, 500 mL, and 1 L. Pipets are available that measure volumes between 0.1 mL and 50 mL. Shown from left to right are 25-, 10-, 5-, and 1-mL pipets.

6. Using Pipets Picking and Using the Appropriate Micropipet • Measuring units smaller than 10 mL requires a pipet. • “Never mouth pipet!” • P-100 or P-200 micropipet • P-10 or P-20 micropipet • P-1000 micropipet Using Micropipets • When measuring tiny volumes, less that 1 mL, a micropipet is used. • A micropipet has four parts • Plunger button • Ejector button • Volume display • Dispensing tip

7. Labeled micropipet. Learning to use each part of a micropipet correctly is essential. On the micropipet shown, the plunger has two “stops.” Pressing to the first stop evacuates air to the volume in the display. Pressing to the second stop evacuates that volume plus another 50% or so. To ensure accurate measurement, feel the difference between the first and second stop before using the pipet. Inaccurate measurement could waste costly reagents and cause invalid experiment results. P-100 Micropipet. This micropipet will measure volumes as small as 10 mL and has precision to 0.2 mL.

8. P-10 Micropipet. P-10 micropipets are common in biotechnology labs. A P-10 micropipet will measure volumes as small as 0.5 mL and has precision to 0.02 mL. A P-10 uses tiny tips that are usually white. P-1000 Micropipet. A P-1000 micropipet will measure up to 1000 mL, or 1 mL, and uses large tips that are usually blue or white in color.

9. A multichannel pipet allows several samples to be measured at the same time, a feature that saves time during an experiment with multiple replications and repetitive pipeting.

10. Vocabulary • Volume – a measurement of the amount of space something occupies • Mass – amount of matter (atoms and molecules) an object contains • Liter – abbreviated “L”; a unit of measure for volume, approximately equal to a quart • Milliliter – abbreviated “mL”; a unit of measure for volume; one one-thousandth of a liter (0.001 L) or about equal to one-half teaspoon • Microliter – abbreviate “mL”; a unit of measure for volume; equivalent to one-thousandth of a milliliter or about the size of the tiniest teardrop • Graduated cylinder – a plastic tube with marks (or graduations) equally spaced to show volumes; measurements are made at the bottom of the meniscus, the lowest part of the concave surface of the liquid in the cylinder • Pipet – an instrument usually used to measure volumes between 0.1 mL and 50 mL • Micropipet – an instrument used to measure very tiny volumes, usually less than a milliliter • Unit of measurement – the form in which something is measured (g, mg, mg, L, mL, mL, km, cm, etc.) • Conversion factor – a number (a fraction) where the numerator and denominator are equal to the same amount; commonly used to convert from one unit to another • Metrics conversion table – a chart that shows how one unit of measure relates to another (for example, how many milliliters are in a liter)

11. 3.1 Review Questions • What instrument would you use to measure and dispense the following volumes? Pick the instrument that is likely to give you the least error for each measurement. • 23.5 mL 6.5 mL 125 mL 7mL 2.87 mL 555mL • Convert the following units to the requested unit: • 1.7 L = _____ mL 235.1 mL = _____ mL 2.37 mL = _____ mL • What numbers should be dialed into a P-10 display if a volume of 3.7 mL is to be measured? • What instrument should be used if a technician wants to fill 40 sets of 16 tubes all with identical volumes?

12. 3.2 Making Solutions • Solution preparation is one of the most essential skills of a biotechnology lab employee. • Solutions are mixtures in which one or more substances are dissolved in another substance. • Solid solutes are measured on balances or scales. • Concentration is measured in several ways: • Mass/volume • Volume/volume • % mass/volume • Molarity • Normality

13. Most analytical balances measure down to milligrams, even though they usually report in grams.

14. Vocabulary • Positive displacement micropipet – an instrument that is generally used to pipet small volumes of viscous (thick) fluids • Solution – a mixture of two or more substances where one (solute) completely dissolves in the other (solvent) • Aqueous – describing a solution in which the solvent is water • Solute – the substance in a solution that is being dissolved • Balance – an instrument that measures mass • Weight – the force exerted on something by gravity; at sea level, it is considered equal to the mass of an object • Gram – abbreviated “g”; the standard unit of mass, approximately equal to the mass of a small paper clip • Solvent – the substance that dissolves the solute • Molarity – a measure of concentration that represents the number of moles of a solute in a liter of solution (or some fraction of that unit) • Normality – a measurement of concentration generally used for acids and bases that is expressed in gram equivalent weights of solute per liter of solution; represents the amount of ionization of an acid or base

15. 3.2 Review Questions • What instrument should be used to measure and dispense the following solutes? Choose the instrument that is likely to give you the least error for each measurement. • 3.5 g of salt 6.5 mg of DNA 12.5 g of gelatin • What is the relation of solute to solvent as a solution becomes more concentrated? • Which of the following are concentration units? • mi/hr g/mL mM°F/°C • 4. Describe how glassware should be prepared before using it to prepare or store solutions.

16. 3.3 Solutions of Given Mass/Volume Concentrations Mass/Volume Solution. Solvent is added until a volume of 10 mL is reached. A protein solution that has a concentration of 1 mg/mL is considered fairly concentrated.

17. Making Mass/Volume Solutions Concentration in Mass/Volume Units ____ g/mL X ____ mL = ____ g of solution concentration volume to be weighed out, desired desired dissolved in the solvent

18. 3.3 Review Questions • Which of the following are mass/volume concentration units? • mg/mL g/mg L/mg mg/mL g/l • What mass of the protein, gelatin, is needed to make 0.5 L of a 3 g/L gelatin solution? • What mass of sugar is need to make 25 mL of a 25 mg/mL sugar solution? • What mass of salt is needed to make 150 mL of a 100 mg/mL salt solution? Describe how the solution is prepared.

19. 3.4 Solutions of Differing % Mass/Volume Concentrations • A percentage represents something that is part of 100. Concentration % Mass/Volume Equation ____ % = ____ percent value decimal value of the g/mL ____ X ____ = ____ g of solute to decimal total volume be measured and value desired (mL) added to the volume desired of solvent

20. Vocabulary • Percentage – a proportion of something out of 100 parts, expressed as a whole number

21. 3.4 Review Questions • What is the decimal equivalent of the following percentages? • 10% 15% 25% 2% 1.5% 0.5% • What mass of gelatin (a protein) is needed to make 0.5 L of a 3% gelatin solution? • What mass of sugar is needed to make 25 mL of a 2.5% sugar solution? • What mass of salt is needed to make 150 mL of a 10% salt solution? Describe how the solution is prepared.

22. 3.5 Solutions of Differing Molar Concentrations Molarity Concentration Equation volume molarity molecular the number of grams to be wanted (L) X desired X weight of the = dissolved in solvent, up to (mol/L) solute (g/mol) the total volume of solution desired

23. Periodic Table. The Period Table of Elements shows the elements (atoms) found in compounds (molecules). Each element is listed along with the atomic weight (mass) of each atom in the element. A NaCl molecule has a molecular weight of about 58.4 atu (atomic mass units) because the Na atom weighs about 23 amu, and the Cl atom weighs about 35.4 amu. Together, in the NaCl molecule, the atoms total approximately 58.4 amu. The mass of a hydrogen atom equals 1 amu.

24. This instrument is a mass spectrometer. Scientists use it to determine the molecular weight of a compound. A “mass spec” can also determine if a sample is contaminated with molecules of different molecular weights.

25. Vocabulary • Mole – the mass, in grams, of 6 X 1023 atoms of molecules of a given substance; one mole is equivalent to the molecular weight of a given substance, reported in grams • Molecular weight – the sum of all the atomic weights of the atoms in a given molecule • amu – abbreviation of atomic mass unit; the mass of a single hydrogen atom • Mass spectrometer – an instrument that is used to determine the molecular weight of a compound

26. 3.5 Review Questions • What is the molecular weight of each of the following compounds? • NaOH MgCl2 MgO HCl • What mass of NaCl is needed for 0.5 L of a 0.5 M NaCl solution? • What mass of MgO is needed for 200 mL of a .025 M MgO solution? • What mass of sodium hydroxide (NaOH) is needed to make 750 mL of a 125 mM NaOH solution? Describe how to prepare the solution.

27. 3.6 Dilutes of Concentrated Solutions Concentrating 1 L Solution. Many chemical and biological reagents are purchased in concentrated form. Concentrated solutions can be prepared initially with a greater amount of solute to solvent, or a solution can be concentrated by removing water. A diluted solution can be prepared by adding solvent to the solution.

28. Diluting a 100 mg/mL Stock Solution to 1 mg/mL.

29. Vocabulary • Dilution – the process in which solvent is added to make a solution less concentrated • Stock solution – a concentrated form of a reagent that is often diluted to form a “working solution” • Buffer – a solution that acts to resist a change in pH when the hydrogen ion concentration is changed • TRIS – a complex organic molecule used to maintain the pH of a solution • TAE buffer – a buffer that is often used for running DNA samples on agarose gels in horizontal gel boxes; contains TRIS, EDTA, and acetic acid

30. 3.6 Review Questions • How do you prepare 40 mL of a 2 mg/mL protein solution from 10 mg/mL protein solution? • How do you prepare 200 mL of 2X enzyme buffer from 10X enzyme buffer solution? • How do you prepare 500 mL of 10 mM NaCl solution from 5 mM NaCl solution? • How do you prepare 3 L of 1X TAE buffer from 50X TAE buffer stock solution?

31. Questions and Comments?